We propose to develop a program to study tolerance to non-MHC tissue specific antigens. Such antigens are among the most relevant self antigens clinically, since they are the putative targets of autoimmune syndromes such as type 1 diabetes mellitus and multiple sclerosis. An understanding of the conditions under which the immune system recognizes self tissue specific antigens is critically important to the determination of how autoimmune responses to such antigens is either initiated or prevented. Toward that end, we have developed a transgenic mouse model with tissue specific expression of a non-MHC antigen. Using a hybrid transgene construct, we have developed a transgenic mouse model with tissue specific expression of a non-MHC antigen. Using a hybrid transgene construct, we have targeted the expression of the influenza hemagglutinin (HA) specifically to the endocrine islet beta cells of the pancreas (Ins-HA transgenic mice). The HA antigen has extremely well defined immunological and biochemical characteristics, enabling detailed studies of the immune response among the CD4 and CD8 subsets of T cells, and among B cells. Our preliminary studies indicate that CD4 and CD8 T cells have been at least partially tolerized to the beta cell specific HA. To identify the mechanisms involved in the induction of tolerance in these mice, we propose three complementary approaches: 1.Presentation of the HA toleragen: We aim to analyze the mechanisms of presentation of the islet specific HA for the induction of tolerance. The critical issues to be addressed here concern the nature of the antigen presenting cells (APC) necessary for the induction of peripheral tolerance. In the case of class I restricted CD8+ T cells, it is possible that encounter with HA antigens directly presented by islet beta cells is a toleragenic signal. However, there is clear precedent for class I presentation of exogenously derived antigen in several in vivo situations. This presents the possibility that beta cell derived HA may also be presented by other APC to deliver a toleragenic signal. In the case of class II restricted CD4+ T cells, the beta cell cannot present HA to CD4+ T cells under normal conditions, so here too it must be determined whether bone marrow derived APC are necessary for the presentation of peripheral antigens in the induction of tolerance. Candidates for such a cell include macrophages, dendritic cells, and B cells. Since our preliminary studies suggest that HA specific B cells were not tolerized in Ins-HA mice, we will address the issue of whether antigen specific B cells may be most effective in presenting tissue specific antigens for tolerance induction. 2.Origins of HA responsive cells: We will undertake an analysis of how each component of the immune system has been affected by the islet specific transgene antigen. Our attention will be directed at several important issues in the induction of peripheral tolerance. For example, autoreactive T cells can be demonstrated even in "tolerant" animals; have these autoreactive cells simply escaped tolerance induction by virtue of low affinity, or do they represent recent thymic emigrants that have not yet been tolerized? Moreover, when tolerance is "broken" to cause autoimmunity, which cells participate in the autoimmune response? 3.T cell receptor affinity and tolerance in T cell receptor transgenic mice: To allow a more precise analysis of the physiology of high versus low affinity HA reactive T cells in Ins-HA mice, we plan to generate T cell receptor (TCR) transgenic mice cloned from CD4 T cell clones with demonstrably high or low affinities for the same HA peptide. Using adoptive transfer of TCR transgenic T cells, we will then be able to follow cells with known receptor specificity, and determine whether clonal deletion, clonal anergy, or clonal exhaustion is induced in different cells on the basis of their receptor affinity.